The present invention relates to DNA molecules encoding splice variants of the melanocortin-1 receptor (MC-R1) protein belonging to the rhodopsin sub-family of G-protein coupled receptors, recombinant vectors comprising DNA molecules encoding MC-R1 splice variant proteins, recombinant host cells which contain a recombinant vector encoding MC-R1 splice variants, the human MC-R1 proteins encoded by the DNA molecule, and methods of identifying selective agonists and antagonists of MC-R1 splice variant proteins disclosed throughout this specification.
Melanocortin receptors belong to the rhodopsin sub-family of G-protein coupled receptors (GPCR""s). Five different subtypes are known. These melanocortin receptors bind and are activated by peptides such as xcex1-, xcex2-, or xcex3-melanocyte stimulating hormones (xcex1-, xcex2-, xcex3-MSH) derived from the pro-opiomelanocortin (POMC) gene. A wide range of physiological functions are believed to be mediated by melanocortin peptides and their receptors.
U.S. Pat. No. 5,532,347, issued on Jul. 2, 1996, to Cone and Mountjoy discloses and claims human and mouse DNA molecules which encode MC-R1 (also known in the art as xcex1-MSH-R). The expressed human protein contains 317 amino acids.
U.S. Pat. No. 5,849,871, issued on Dec. 15, 1998, to Cone and Mountjoy discloses and claims human and mouse MC-R1. As noted in the previous paragraph, the expressed human protein contains 317 amino acid residues.
Mountjoy, et al. (1992, Science 257: 1248-1251) describe DNA molecules and the concomitant protein for human MC-R1 and human MC-R2.
Chhajlani, et al. (1992, FEBS Letters 309: 417-420) also disclose a human DNA molecule comprising an open reading frame which encodes human MC-R1.
Cone et al. (1996, Recent Progress in Hormone Research 51: 287-318) reviews the state of known mammalian melanocortin receptors, from MC-R1 through MC-R5.
Jackson (1997, Human Molecular Genetics 6: 1613-24) and Koppula, et al. (1997, Human Mutation 9:30-36) review the occurrence and potential significance of polymorphisms within the coding sequence of the human MC-R1 form A.
It is desirable to correlate in vivo data with in vitro biochemical activity of compounds.
It is also desirable to select compounds which activate one or more human melanocortin receptor proteins in vitro.
It is further desirable to discover new drugs which effect pathophysiological processes by modulating melanocortin receptor activity, followed by human clinical trials.
The present invention addresses and meets these needs by disclosing isolated nucleic acid molecules which express splice variants of human MC-R1, recombinant vectors which house these nucleic acid molecules, recombinant host cells which expresses these alternative forms of human MC-R1 and/or biologically active equivalents, and pharmacological properties of these human MC-R1 proteins.
The present invention relates to a series of isolated nucleic acid molecules (polynucleotides) which encode novel variants of the human melanocortin-1 receptor protein, referred to herein as MC-R1B proteins. The nucleic acid molecules of the present invention are substantially free from other nucleic acids. These isolated nucleic acid molecules encode a MC-R1 protein which contains an intracellular domain with an additional 65 amino acid residues in comparison to the previously disclosed human MC-R1, referred to herein also as MC-R1A. Therefore, the present invention relates to isolated nucleic acid molecules (polynucleotides) which encode a mRNA which expresses a novel human MC-R1 protein, these DNA molecules including but by no means being limited to DNA molecules comprising the nucleotide sequence disclosed herein as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, and SEQ ID NO:25.
The present invention also relates to isolated nucleic acid molecules which represent human genomic clones which comprise at least a single intron within the open reading frame which encodes novel variants of human MC-R1B protein. Therefore, the present invention relates to isolated nucleic acid molecules (polynucleotides) which encode a RNA molecule which is spliced to generate a mRNA molecule which encodes a novel human MC-R1 protein variant, these DNA molecules including but by no means being limited to DNA molecules comprising the nucleotide sequence disclosed herein as SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24. To this end, the present invention also relates to the respective mRNA molecule generated from each of the DNA molecules depicted as SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24.
The isolated nucleic acid molecules of the present invention comprise a 3xe2x80x2 extension in the open reading frame which encodes a 65 amino acid COOH-terminal extension to known MC-R1. Therefore, the present invention relates to isolated nucleic acid molecules, both DNA and RNA molecules, that encode for a splice variant of known MC-R1 which encodes for this 65 amino acid COOH-terminal extension. The totality of nucleic acid molecules of the present invention, including genomic DNA, cDNA, RNA and mRNA, will be referred to herein as xe2x80x9cMC-R1 splice variantsxe2x80x9d, which will identify a disclosed nucleic acid molecule which encodes an protein with melanocortin 1 receptor activity in combination with this additional 3xe2x80x2 exon which encodes a 65 amino acid COOH-terminal extension. These isolated nucleic acid molecules include but are by no means limited to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18; SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:25.
The present invention also relates to biologically active fragments or mutants of MC-R1 splice variants which encodes mRNA expressing a novel human MC-R1. Any such biologically active fragment and/or mutant of the MC-R1 splice variants disclosed herein will encode either a protein or protein fragment which at least substantially mimics the pharmacological properties of a wild-type MC-R1 protein and comprises at least a portion of the COOH terminal amino acid extension disclosed as SEQ ID NO:27. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for MC-R1B function.
A preferred aspect of this portion of the present invention is set forth as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18; SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:25, human nucleic acid molecules which comprise the complete open reading frame for the MC-R1B proteins of the present invention.
The isolated nucleic acid molecules of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification, including but not limited to the isolated nucleic acid molecules as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18; SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:25.
The present invention also relates to subcellular membrane fractions of the recombinant host cells (both prokaryotic and eukaryotic as well as both stably and transiently transformed cells) which contain the proteins encoded by the nucleic acids of the present invention. These subcellular membrane fractions will comprise either wild-type or mutant forms of the human melanocortin-1 receptor proteins which comprise the COOH-terminal extension at levels substantially above endogenous levels and hence will be useful in various assays described throughout this specification.
The present invention also relates to a substantially purified form of the COOH-terminal variants of human melanocortin-1 receptor protein, which comprises the amino acid sequences as disclosed in FIGS. 5A-5F and as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26. These MC-R1 proteins comprise a 65 amino acid extension at the COOH-terminus when compared to known human MC-R1 and are referred to throughout this specification as MC-R1B proteins or MC-R1 splice variant proteins.
The present invention also relates to biologically active fragments and/or mutants of the human MC-R1B proteins disclosed throughout this specification, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for MC-R1B function.
The present invention also relates to isolated nucleic acid molecules which are fusion constructions expressing fusion proteins useful in assays to identify compounds which modulate wild-type vertebrate MC-R1B activity. A preferred aspect of this portion of the invention includes, but is not limited to, glutathione S-transferase (GST)-MC-R1B fusion constructs which include, but are not limited to, either the intracellular domain of human MC-1RB as an in-frame fusion at the carboxy terminus of the GST gene, or the extracellular and transmembrane ligand binding domain of MC-R1B fused to the amino terminus of GST, or the extracellular and transmembrane domain of MC-R1B fused to an immunoglobulin gene by methods known to one of ordinary skill in the art. Soluble recombinant GST-MC-R1B fusion proteins may be expressed in various expression systems, including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using a baculovirus expression vector (pAcG2T, Pharmingen).
Therefore, the present invention relates to methods of expressing the human MC-R1B proteins disclosed herein and biological equivalents, assays employing these gene products, recombinant host cells which comprise DNA constructs which express these receptor proteins, and compounds identified through these assays which act as agonists or antagonists of MC-R1B activity.
The present invention also relates to polyclonal and monoclonal antibodies raised in response to either the human form of a MC-R1B protein, or a biologically active fragment thereof.
It is an object of the present invention to provide an isolated nucleic acid molecule which encodes a novel form of human MC-R1B, or human MC-R1B fragments, mutants or derivatives of the human MC-R1B proteins as set forth in FIGS. 5A-5F and SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, aminoterminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for vertebrate MC-R1B function.
It is a further object of the present invention to provide the human MC-R1B proteins or protein fragments encoded by the nucleic acid molecules referred to in the preceding paragraph.
It is a further object of the present invention to provide recombinant vectors and recombinant host cells which comprise a nucleic acid sequence encoding these human MC-R1B proteins or biological equivalents thereof.
It is an object of the present invention to provide a substantially purified form of any of the human MC-R1B proteins, including but not limited to the proteins as set forth in FIGS. 5A-5F and SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26.
It is an object of the present invention to provide for biologically active fragments and/or mutants of the human MC-R1B proteins disclosed herein, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use.
It is also an object of the present invention to provide for MC-R1B-based assays to select for modulators of this receptor protein. These assays are preferably cell based assays whereby a DNA molecule encoding MC-R1B is transfected or transformed into a host cell tested wherein this recombinant host cell is allowed to grow for a time sufficient to express MC-R1B prior to use in various assays described herein.
Alternatively, an assay utilizing substantially purified membrane fractions from such a transfected host cell with a DNA vector encoding the MC-R1B protein, such that binding of test compounds in relation to a known MC-R1B ligand may be tested. To this end, it is a further object to provide for membrane preparations from host cells transfected or transformed with a DNA molecule encoding MC-R1B for use in assays to select for modulators of MC-R1B activity.
It is also an object of the present invention to provide for MC-R1B-based in-frame fussion constructions, methods of expressing these fusion constructs, biological equivalents disclosed herein, related assays, recombinant cells expressing these constructs, and agonistic and/or antagonistic compounds identified through the use of the nucleic acid encoding vertebrate MC-R1B protein as well as the expressed protein.
As used herein, xe2x80x9cMC-R1 splice variantsxe2x80x9d and/or xe2x80x9cMC-R1B splice variantsxe2x80x9d, refers to a nucleic acid molecule which encodes a protein with melanocortin-1 receptor activity which comprises a 3xe2x80x2 exon segment which encodes a 65 amino acid COOH-terminal extension identified in SEQ ID NO:27. Such nucleic acid molecules include but are not limited to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18; SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:25.
As used herein, xe2x80x9cMC-R1Bxe2x80x9d and/or xe2x80x9cMC-R1 splice variant proteinsxe2x80x9d refers to the proteins translated from the MC-R1 splice variant nucleic acid molecules disclosed herein. These human MC-R1B proteins include but are not limited to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26.
As used herein, xe2x80x9cGPCRxe2x80x9d refers toxe2x80x94G-protein coupled receptorxe2x80x94.
The terms xe2x80x9cisolatedxe2x80x9d and xe2x80x9cpurifiedxe2x80x9d are used interchangeably to denote a nucleic acid, protein, membrane fraction and such which is substantially free from other like components.
Whenever used herein, the term xe2x80x9cmammalian hostxe2x80x9d will refer to any mammal, including a human being.